# Patent

## Title
Spatially-defined macrocyclic compounds useful for drug discovery

## Abstract

Novel spatially-defined macrocyclic compounds containing specific conformational control elements are disclosed. Libraries of these macrocycles are then used to select one or more macrocycle species that exhibit a specific interaction with a particular biological target. In particular, compounds according to the invention are disclosed as agonists or antagonists of a mammalian motilin receptor and a mammalian ghrelin receptor.

## Background
RELATED APPLICATION INFORMATION
This application is a continuation under 35 U.S.C. §120 of U.S. patent application Ser. No. 12/197,610, filed Aug. 25, 2008 and issued as U.S. Pat. No. 8,022,252, which is a divisional application of U.S. patent application Ser. No. 10/911,221, filed Aug. 2, 2004 and issued as U.S. Pat. No. 7,452,862, which claims the benefit of U.S. Patent Application Ser. No. 60/491,248, filed Jul. 31, 2003. The disclosure of each application is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
This invention relates to spatially-defined macrocyclic compounds with specific conformational control elements. It also relates to the generation of libraries of these macrocycles. These libraries are then used to select one or more macrocycle species that exhibit a specific interaction with a particular biological target.
BACKGROUND OF THE INVENTION
Among the variety of compounds that have consistently been found to possess potent and selective biological activity are natural products and peptides. Indeed, members of these classes have become useful pharmaceutical agents. Unfortunately, each type has limitations that have restricted the wider utility of these structures.
In fact, natural products often have extremely complex structures that are difficult to synthesize, particularly in the combinatorial fashion that would provide access to a greater number of analogues with which to define pharmacophoric elements and best explore modulation of the biological properties of the parent compound. Nevertheless, some efforts have been successful at constructing natural product libraries containing a modest number of analogues.
Peptides, on the other hand, have been at the forefront of the development of combinatorial chemistry due to their ease of synthesis on solid support, the reproducible and high-yielding reactions involved, and the ready availability of starting materials. Peptides being the endogenous ligands for a number of enzymes and receptors, their modification can be performed to develop even more potent agonists or inhibitors of these same receptors and enzymes. In addition, combinatorial peptide libraries have been used to find a number of previously unknown active sequences for a wide array of enzyme and receptor systems. However, peptidic compounds are plagued by the usual limitations associated with the direct use of peptides as pharmaceuticals, including rapid metabolic degradation by proteases, short pharmacokinetic half-life, difficulty in transport to site of action in tissues and organs, poor oral bioavailability and solubility, potential antigenicity, as well as high manufacturing costs.
Nevertheless, the densely functionalized and structurally diverse nature of peptides is advantageous when seeking new drug molecules. Hence, peptides are primarily used as the starting point or template for the development of new pharmaceutical leads that often results in structures that only partially resemble, if at all, the initial active peptide. In particular, the recognition potential of the amino acid side chains has resulted in attempts to incorporate these side chains into non-peptidic rigid scaffolds that attempt to duplicate the conformational display required for optimal interaction between the molecule and the target, as well as mimic standard protein and peptide secondary structural elements. For example, sugars and aromatic rings have been exploited as rigid scaffolds containing amino acids or analogues as pendant moieties at one or more positions. Compounds and combinatorial libraries utilizing 3- and 4-substituted pyrrolidines as a central template for display of interacting functionality have been disclosed in U.S. Pat. No. 5,646,285 and U.S. Pat. No. 5,891,737.
In another approach, cyclic structures can greatly improve the pharmacological and pharmacokinetic profiles of peptides (Molecular Diversity 2000 (pub. 2002), 5, 289-304). Cyclic peptides analogues offer a number of benefits compared with the corresponding linear analogues, including restricted conformational mobility, defined topology, enhanced stability to proteolytic enzymes and modified polarity.
Furthermore, cyclic peptides can enhance potency, selectivity, stability, bioavailability and membrane permeability. The stability to enzymatic degradation of the cyclic structure arises from the difficulty of such molecules to attain the extended conformation required to be recognized as a substrate for peptidases. Very large mixture libraries (108 members or more) of cyclic peptides have been described in WO 98/54577.
However, larger rings are often too flexible and can occupy too many conformations to be useful. Further, their molecular size and resulting physicochemical characteristics do not fit the typical requirements for being “drug-like.” Small cyclic peptides containing the key interacting residues would provide the necessary conformational restriction, but may have other disadvantages, including synthetic difficulty, ease of dimerization, unfavorable ring strain caused by the presence of the preferred trans amide bonds, lack of stability towards metabolism and hydrolysis to release that strain and limited topological diversity.
Most attention in combinatorial chemistry has been devoted to producing diversity in terms of chemical composition. However, essentially no effort has been directed at integrating this with diversity in terms of the crucial three-dimensional structure.
The use of certain tether elements to control conformation was reported in WO 01/25257. However, although those tethers were successful in restricting the conformational display of the molecule, they only were able to duplicate a portion of the spatial region accessible to a linear molecule, which can contain hundreds if not thousands of possible conformations. To better cover the available conformational space, additional tether elements that define new conformations are required. In addition, the tethers in the previous report were generally hydrophobic in nature. This effects key properties of the macrocyclic molecules such as solubility and log P that are known to have an impact on the compound's pharmacological properties, in particular oral bioavailability. Further, variation of these physicochemical properties is often required in order to optimize the desired characteristic of a molecule as a therapeutic agent. As well, the early tethers were rather limited in their chemical functionality. Since this part of the molecule also could have interactions with a biological target in addition to its conformational control function, a greater diversity in the chemical functional groups could prove advantageous. The more chemically diverse tethers of the present invention therefore have been designed to address these limitations of the existing art and provide the following benefits:

    
    
        Access to previously inaccessible conformations
        Modification of physicochemical parameters
        Improvement of pharmacokinetic profile
        Additional interacting functionalities for modulation of biological activity
    
    


Growing evidence suggests that molecular rigidity confers favorable pharmacokinetic properties on molecules and leads to improved clinical success (J. Med. Chem. 2003, 46, 1250-1256; J. Med. Chem. 2002, 45, 2615-2623). The tethers of the present invention therefore will be extremely useful in utilizing these macrocyclic molecules in the search for new pharmaceuticals. Examples of the activity that have been exhibited by representative molecules of the invention are provided.
Therefore, there remains a need for specifically designed chemical entities built on a macrocyclic framework, which exploit the three-dimensional conformation changes triggered by peptidic modifications and/or by inserting specific tether-like portions, in their macrocyclic skeleton.
SUMMARY OF THE INVENTION
The present invention is directed towards spatially-defined macrocyclic compounds which incorporate conformational control elements in order to limit their three-dimensional structure to a small number of spatial orientations. These compounds are defined by general formula (1):




wherein
A1, A2, A3 and A4 are natural amino acid residues or unnatural amino acid residues;
A3 and A4 are optionally present;
W is O or —NR1—, wherein R1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl and sulfonyl;
T is a bivalent radical chosen from the group consisting of










wherein q1, q2, q3, q6, q7, q8, q9, q10, q11, q13, q15 and q16 are each independently 1, 2 3, 4 or 5;
q4 and q18 are independently 1 or 2;
q5 is 2, 3 or 5;
q12 and q14 are each independently 0, 1, 2, 3 or 4;
q17 is 0, 1, 2 or 3;
P1, P2, P3 P4 and P5 are each independently O, S or NH;
P6 is N or CH;
P7 is O or CR52R53;
R36 is hydrogen, C1-C6 alkyl, benzyl or acyl;
R50 and R51 are independently selected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy, or amino with the proviso that if one of R50 or R51 is hydroxy, alkoxy or amino, the other is hydrogen or alkyl;
R52 and R53 are independently selected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy, or amino with the proviso that if one of R52 or R53 is hydroxyl, alkoxy or amino, the other is hydrogen or alkyl;
R54, R55, R56, R57 and R58 are independently selected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy, or amino;
RAA is a side-chain of a natural amino acid or a side-chain of an unnatural amino acid;
(W) indicates the point of attachment of T to W;
and (A1) indicates the point of attachment of T to A1.
Libraries of these compounds are then used to select one or more macrocycle species that exhibit a specific interaction with a particular biological target. Such targets include, but are not limited to, enzymes and receptors. More particularly, the macrocyclic libraries of the invention serve as a readily accessible source of diverse macrocyclic compounds for use in identifying new biologically active macrocyclic compounds through pharmaceutical candidate screening assays, for use in studies defining structure/activity relationships, and/or for use in clinical investigation.
In particular, compounds of formula (I) are disclosed as agonists or antagonists of a mammalian motilin receptor and a mammalian ghrelin receptor.
While the invention will be described in conjunction with an example embodiment, it will be understood that it is not intended to limit the scope of the invention to such embodiment. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included as defined by the appended claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a general scheme showing one approach to the solid phase synthesis of compounds of the invention.
FIG. 2 is a general scheme showing a second approach to the solid phase synthesis of compounds of the invention.
FIGS. 3-19 are synthetic schemes that show routes to specific tethers (T) used for the synthesis of compounds of the invention.



DETAILED DESCRIPTION OF THE INVENTION
The macrocyclic compounds of the present invention incorporate a variety of tethers, thus allowing coverage of a specific section of conformational space. Furthermore, these tethers are selected on the basis of their ability to synthetically produce macrocycles in reasonable yield across a wide range of sequences. Accordingly, the compounds of the invention, which incorporate these tethers, represent a wide variety of different conformations, with some more rigid and others more flexible. In addition, some of the tethers are much more rigid in their conformation, sometimes displaying essentially only one low energy form. In these cases, improved biological results would provide excellent information on the specific, optimum bioactive conformation. Additionally, in contrast to many traditional approaches, the same synthetic routes and methods are employed in this optimization process. The ability to rapidly access such information transforms what is usually an extremely difficult and time intensive task into a much more straight forward undertaking.
As such, this invention permits the simultaneous investigation of chemical and conformational diversity within a single structural framework and therefore possesses great potential for use in increasing the speed and efficiency of research aimed at new pharmaceuticals.
Accordingly, the invention provides macrocyclic compounds of formula (I) wherein A1, A2, A3, A4, W and T are as defined previously.




In a specific embodiment, there are provided compounds of formula (I), T is chosen from the following bivalent radicals:













wherein Y is selected from hydrogen, alkyl, benzyl or acyl.
The invention also provides compounds of formula 1 wherein at least one of A1, A2, A3 and A4 can further be a protected natural or unnatural amino acid residue.
The present invention has applicability to a broad range of biological targets that likewise represent diverse therapeutic indications. Active compounds initially generated can be further optimized and refined to eventually provide lead clinical candidates. A further advantage of the invention is that these subsequent steps in the optimization process can be conducted utilizing the same basic chemical synthesis pathway, hence greatly simplifying and speeding up what is typically an extremely time-consuming phase of the overall drug discovery process.
In particular, the invention provides compounds of formula (I)) which are agonists or antagonists of a mammalian motilin receptor and/or a mammalian ghrelin receptor.
Motilin, a linear 22-amino acid peptide, plays a critical regulatory role in the GI physiological system through governing of fasting gastrointestinal motor activity. As such, the peptide is periodically released from the duodenal mucosa during fasting in mammals, including humans. More precisely, motilin exerts a powerful effect on gastric motility through the contraction of gastrointestinal smooth muscle to stimulate gastric emptying, decrease intestinal transit time and initiate phase III of the migrating motor complex in the small bowel. Due to the critical and direct involvement of motilin in control of gastric motility, agents that either diminish (hypomotility) or enhance (hypermotility) the activity at the motilin receptor, are a particularly attractive area for further investigation in the search for new effective pharmaceuticals towards these indications. Macrocyclic antagonists of the motilin receptor are disclosed in U.S. Prov. Pat. Appl. Ser. No. 60/479,223.
Likewise, ghrelin is a key peptide hormone involved in a number of important physiological functions including growth hormone secretion, maintenance of energy balance, appetite and gut motility. As such, antagonists of this receptor have been investigated for treatment of obesity, while ghrelin agonists have interest in treatment of a variety of diseases, including conditions caused by growth hormone deficiency, wasting syndrome, and GI disorders involving dysmotility.

    
    
        Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln motilin (human, porcine, SEQ ID NO:1)
        Gly-Ser-Ser(Oct)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Vd-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg ghrelin (human SEQ ID NO:2)
    
    


EXAMPLES
Synthesis Method
An assortment of synthetic strategies, involving both solution and solid phase techniques, can be used to access the macrocyclic compounds of the invention, several of which have already been disclosed in WO 01/25257.
An outline of a first approach to the solid phase synthesis of the compounds of the invention, using a thioester linker strategy is provided in Figure (I). A second approach, called ring-closing metathesis (RCM), is also generally outlined in figure (II).
In both, the construction involves four phases: first is synthesis of the building blocks, comprising mainly recognition elements for interaction at biological targets, plus the key tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations and those described in the Standard Procedures herein. The precursors from the assembly are then cyclized in the third stage, which could involve multiple steps, to provide the macrocyclic structures. Finally, a post-cyclization processing stage involving removal of protecting groups and optional purification then provides the desired final compounds.
General Information
Reagents and solvents were of reagent quality or better and were used as obtained from various commercial suppliers unless otherwise noted. DMF, DCM, DME and THF used are of DriSolv® (EM Science, now EMD Chemicals, Inc., part of Merck KGaA, Darmstadt, Germany) or synthesis grade quality except for (i) deprotection, (ii) resin capping reactions and (iii) washing. NMP used for the amino acid (AA) coupling reactions is of analytical grade. DMF was adequately degassed by placing under vacuum for a minimum of 30 min prior to use. Boc- and Fmoc-protected amino acids and side chain protected derivatives, including those of N-methyl and unnatural amino acids, were obtained from commercial suppliers or synthesized through standard methodologies known to those in the art. Ddz-amino acids were either synthesized by standard methods, or obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (Louisville, Ky., USA). Bts-amino acids were synthesized by established procedures. Hydroxy acids were obtained from commercial suppliers or synthesized from the corresponding amino acids as described in the literature (Tetrahedron 1989, 45, 1639-1646; Tetrahedron 1990, 46, 6623-6632; J. Org. Chem. 1992, 57, 6239-6256.; J. Am. Chem. Soc. 1999, 121, 6197-6205). Analytical TLC was performed on pre-coated plates of silica gel 60F254 (0.25 mm thickness) containing a fluorescent indicator.
1H and 13C NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer (Varian, Inc., Palo Alto, Calif.) and are referenced internally with respect to the residual proton signals of the solvent. Information about the conformation of the molecules in solution can be determined utilizing appropriate two-dimensional NMR techniques known to those skilled in the art.
HPLC analyses are performed on a Waters Alliance® system 2695 running at 1 mL/min using an Xterra® MS C18 column (or comparable) 4.6×50 mm (3.5 μm). A Waters 996 PDA provided UV data for purity assessment (Waters Corporation, Milford, Mass.). An LCPackings (Dionex Corporation, Sunnyvale, Calif.) splitter (50:40:10) allowed the flow to be separated in three parts. The first part (50%) went to a mass spectrometer (Micromass® Platform II MS equipped with an APCI probe) for identity confirmation. The second part (40%) went to an evaporative light scattering detector (ELSD, Polymer Laboratories, now part of Varian, Inc., Palo Alto, Calif., PL-ELS-1000TM) for purity assessment and the last portion (10%) to a chemiluminescence nitrogen detector (CLND, Antek® Model 8060, Antek Instruments, Houston, Tex., part of Roper Industries, Inc., Duluth, Ga.) for quantitation and purity assessment. Data was captured and processed utilizing the most recent version of the Waters Millennium® software package (Milford, Mass.).
Preparative HPLC purifications were performed on final deprotected macrocycles using the Waters FractionLynx® system, on an XTerra® MS C18 column (or comparable) 19×100 mm (5 μm). The injections were done using an At-Column-Dilution configuration with a Waters 2767 injector/collector and a Waters 515 pump running at 2 mL/min. The mass spectrometer, HPLC, and mass-directed fraction collection are controlled via MassLynx® software version 3.5 with FractionLynx®. Fractions (13×125 mm tubes) shown by MS analysis to contain the product were evaporated under reduced pressure, most typically on a centrifugal evaporator system (Genevac® HT-4 (Genevac Inc, Valley Cottage, N.Y.), ThermoSavant Discovery®, SpeedVac® or comparable (Thermo Electron Corporation, Waltham, Mass.) or, alternatively, lyophilized. Compounds were then thoroughly analyzed by LC-MS-UV-ELSD-CLND analysis for identity confirmation, purity and quantity assessment.
Automated medium pressure chromatographic purifications were performed on an Isco CombiFlash® 16x system with disposable silica or C18 cartridges that permitted up to sixteen (16) samples to be run simultaneously (Teledyne Isco, Inc., Lincoln, Nebr.). MS spectra were recorded on a Waters Micromass® Platform II or ZQTM system. HRMS spectra were recorded with a VG Micromass ZAB-ZF spectrometer. Chemical and biological information were stored and analyzed utilizing the ActivityBase® database software (ID Business Solutions Ltd., Guildford, Surrey, UK).
The term “concentrated/evaporated/removed under reduced pressure” indicates evaporation utilizing a rotary evaporator under either water aspirator pressure or the stronger vacuum provided by a mechanical oil vacuum pump as appropriate for the solvent being removed. “Dry pack” indicates chromatography on silica gel that has not been pre-treated with solvent, generally applied on larger scales for purifications where a large difference in Rf exists between the desired product and any impurities. “Flash chromatography” refers to the method described as such in the literature and is applied to chromatography on silica gel (230-400 mesh, EM Science) used to remove impurities some of which may be close in Rf to the desired material. Methods specific for solid phase chemistry are detailed separately.
General Methods for Solid Phase Chemistry
These methods can be equally well applied for the synthesis of single compounds or small numbers of compounds, as well as for the synthesis of libraries of compounds of the present invention.
For solid phase chemistry, the solvent choice is important not just to solubilize reactants as in solution chemistry, but also to swell the resin. Certain solvents interact differently With the polymer matrix depending on its nature and can affect this swelling property. As an example, polystyrene (with DVB cross-links) swells best in nonpolar solvents such as DCM and toluene, while shrinking when exposed to polar solvents like alcohols. In contrast, other resins such as PEG-grafted ones like TentaGel®, Rapp Polymere GmbH, Tübingen, Germany), maintain their swelling even in polar solvents. For the reactions of the present invention, appropriate choices can be made by one skilled in the art. In general, polystyrene-DVB resins are employed with DMF and DCM common solvents. The volume of the reaction solvent required is generally 1-1.5 mL per 100 mg resin. When the term “appropriate amount of solvent” is used in the synthesis methods, it refers to this quantity. The recommended quantity of solvent roughly amounts to a 0.2 M solution of building blocks (linkers, amino acids, hydroxy acids, and tethers, used at 5 eq relative to the initial loading of the resin). Reaction stoichiometry was determined based upon the “loading” (represents the number of active functional sites, given as mmol/g) of the starting resin.
The reaction can be conducted in any appropriate vessel, for example round bottom flask, solid phase reaction vessel equipped with a fritted filter and stopcock, or Teflon® (DuPont, Wilmington, Del.)-capped jar. The vessel size should be such that there is adequate space for the solvent, and that there is sufficient room for the resin to be effectively agitated taking into account that certain resins can swell significantly when treated with organic solvents. The solvent/resin mixture should fill about 60% of the vessel. Take note that all agitations for solid phase chemistry are best conducted with an orbital shaker (for example Form a® Scientific, model 430, 160-180 rpm) (Thermo Electron Corporation, Waltham, Mass.), except for those where scale makes use of gentle mechanical stirring more suitable, to ensure adequate mixing which is generally accepted to be important for a successful reaction.
The volume of solvent used for the resin wash is a minimum of the same volume as used for the reaction, although more is generally used to ensure complete removal of excess reagents and other soluble residual by-products. Each of the resin washes specified in the Examples should be performed for a duration of at least 5 min with agitation (unless otherwise specified) in the order listed. The number of washings is denoted by “nx” together with the solvent or solution, where n is an integer. In the case of mixed solvent washing systems, both are listed together and denoted solvent 1/solvent 2. The ratio of the solvent mixtures DCM/MeOH and THF/MeOH used in the washing steps is (3:1) in all cases. Other mixed solvents are as listed. After washing, drying in the “standard manner” means that the resin is dried first in air (1 h), and subsequently under vacuum (oil pump usually) until full dryness is attained (minimum 30 min, to O/N).
For representative examples of the new tether moieties disclosed herein, the synthetic routes presented in FIGS. 3-19 are employed with additional information on selected examples presented further below. Although the routes described represent a specific protection strategy, other suitable protecting groups known in the art can also be employed.
Example T12
Standard Procedure for the Synthesis of Tether T12
For an outline of this route, see FIG. 3. In a 3-L flame-dried three-neck flask, a solution of (aminomethyl)phenylthiobenzyl alcohol (12-0, 96 g, 0.39 mol) in degassed DMF (1 L, 0.4 M) was prepared. To this was added Ddz-N3 (0.95 eq), followed by TMG (0.39 mol, 49 mL). The reaction was stirred for 10 min, then DIPEA (68 mL, 0.39 mol) added. The mixture was heated at 50° C. under N2 until TLC indicated no Ddz-N3 remained (48 h typically). (TLC eluent: EtOAc:Hex 50:50; detection: ninhydrin). Upon completion, to the reaction mixture was added 3 L citrate buffer and the separated aqueous layer extracted with Et2O (3×1500 mL). The combined organic phase was washed sequentially with citrate buffer (2×200 mL), water (2×200 mL) and brine (2×200 mL). The organic layer was dried over MgSO4, filtered and the filtrate evaporated under reduced pressure. A dark orange oil was obtained, which was purified by dry-pack. For this procedure, the oil was first dissolved in EtOAc:Hex:DCM:TEA (20:80:1:0.5, v/v/v/v). At this point, a little extra DCM was sometimes required to ensure complete dissolution. The solution was loaded onto the column, then the column eluted with EtOAc:Hex:DCM:Et3N (20:80:1:0.5) until all the impurities were separated out as indicated by TLC, paying particular attention to that closest to the desired product. The elution was then continued with EtOAc:hexanes:Et3N 30:70:0.5 (v/v/v) and finally with EtOAc:hexanes:Et3N (50:50:0.5) to elute the desired product. After removal of the solvent from the fractions containing the product under reduced pressure, the residue was dissolved in the minimum amount of DCM, a three-fold larger volume of hexanes added, then the solvents again evaporated under reduced pressure. This treatment was repeated until an off-white foam was obtained. The latter solidified while drying under vacuum (oil pump). Alternatively, the material yielded a solid after sequential concentration with DCM (1×) and hexanes (2×). Tether T12 was obtained as an off-white solid (85-90% yield).
Example T13
Standard Procedure for the Synthesis of Tether T13
Protected versions of tether T13 are accessed through a route (see FIG. 4) analogous to that described below in more detail for T14, except starting from H-Ser-OEt.HCl, in an overall yield of 14-30% for the 6 step sequence.
1H NMR (CDCl3): δ 7.53 (1H, s, RR′C═CH—O), 6.42-6.58 (2H, m, Ph), 6.30-6.38 (1H, m, Ph), 5.40-5.50 (1H, m, NH), 4.57 (2H, s, CH2OH), 4.40 (2H, d, CH2NHDdz), 3.78 (6H, s, 2×(CH3OPh)), 2.23-2.00 (1H, broad, OH), 1.76 (6H, s, RR′ C(CH3)2).
13C NMR (CDCl3): δ 162, 161, 155, 149, 141, 136, 103, 99, 82, 57, 56, 39, 29.
Example T14
Standard Procedure for the Synthesis of Tether T14
See FIG. 5 for an outline of the synthetic scheme.
Step T14-1:

    
    
        A solution of 4.4 M sodium methoxide in MeOH (1.0 mL, 4.6 mmol, 0.01 eq) in DCM (300 mL) at 0° C. was diluted with MeOH (35 mL). Dichloroacetonitrile (50 g, 455 mmol, 1.0 eq) was added over 45 min and the resulting mixture stirred at 0° C. for 1 h. L-Cysteine ethyl ester hydrochloride (84.5 g, 455 mmol, 1.0 eq) was added and the reactionstirred O/N at rt. The reaction mixture was diluted with DCM and water. The separated aqueous phase was extracted with DCM (2×). The combined organic phase was dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. The crude product obtained was acceptable for use in the next step without further purification.
    
    


Step T14-2:

    
    
        To a solution of the crude product from step T14-1 (455 mmol based on the theoretical yield) in DCM (500 mL) was added DIPEA (119 mL, 652.5 mmol, 1.5 eq). The resulting mixture was stirred at 50° C. for 5 h, then at rt O/N. The reaction was monitored by TLC (30% EtOAc: 70% Hex; detection: UV and CMA, Rf=0.29). Upon completion, the reaction mixture was diluted with DCM and water. The separated aqueous phase was extracted with DCM (2×). The combined organic phase was dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. 1H NMR was used to verify the purity and identity of the intermediate compound. The crude product obtained was acceptable for use in the next step without further purification (yield: 100%).
    
    


Step T14-3:

    
    
        To a solution of the crude product from step T14-2 (77 g, 375 mmol, 1.0 eq) in DMF (500 mL) was added sodium azide (122 g, 1874 mmol, 5.0 eq). The resulting mixture was mechanically stirred at 65° C. O/N. The reaction was monitored by 1H NMR because the starting material and product co-eluted on TLC. After completion and cooling to rt, the reaction mixture was diluted with Et2O and an aqueous solution of saturated NH4Cl. The separated aqueous phase was extracted with Et2O (2×). The combined organic phase was washed with brine, dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. 1H NMR was used to verify the purity and identity of the intermediate compound. The crude product obtained was acceptable for use in the next step without further purification (yield: 93%).
    
    


Step T14-4:

    
    
        To a solution of the crude azide from step T14-3 (73.1 g, 345 mmol, 1.0 eq) in 95% EtOH (700 mL) was added 10% Pd/C (18.3 g, 17.3 mmol, 0.05 eq). Hydrogen gas was bubbled into the suspension for 1 h, then the resulting mixture stirred 0/N with a balloon of hydrogen. The reaction was monitored by TLC (30% EtOAc: 70% Hex; detection: UV and ninhydrin.). The final product remained at the baseline and was positive to ninhydrin. If the reaction was not complete as indicated by TLC, another portion of 10% Pd/C (25% of that originally used) was added, hydrogen bubbled through the solution and the resulting suspension was stirred at rt again O/N. The reaction solution was filtered through a Celite® (World Minerals Inc., Santa Barbara, Calif.) pad and the pad rinsed thoroughly with EtOAc (until no further product was being recovered as indicated by TLC). 1H NMR was used to verify the purity and identity of the intermediate compound. The crude product obtained was acceptable for use in the next step without further purification (yield: 93%).
    
    


Step T14-5:

    
    
        To a solution of the crude amine from step T14-4 (59.5 g, 320 mmol, 1.0 eq) in degassed (maintained on vacuum pump for 1 h) DMF (200 mL) were sequentially added Ddz-N3 (93.3 g, 352 mmol, 1.1 eq), TMG (40.1 mL, 320 mmol, 1.0 eq) and DIPEA (55.8 mL, 320 mmol, 1.0 eq). The resulting solution was stirred at rt for 2 d. The reaction was monitored by TLC (100% EtOAc; detection: UV and ninhydrin, Rf=0.52). Upon completion, the reaction mixture was diluted with Et2O and an aqueous solution of citrate buffer (1 M). The separated aqueous phase was extracted with Et2O (2×). The combined organic phase was washed with citrate buffer (1 M, 2×), water (2×), and brine (2×), then dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. The crude product was purified by dry-pack (20% EtOAc: 80% Hex to 50% EtOAc: 50% Hex) to give the protected amino ester as a yellow solid. 1H NMR was used to verify the identity of the intermediate compound (yield: 65%).
    
    


Step T14-6:

    
    
        To a solution of the protected amino ester from step T14-5 (10.5 g, 25.7 mmol, 1.0 eq) in THF (150 mL) at 0° C. were added lithium borohydride (1.68 g, 77.1 mmol, 3.0 eq) and MeOH (3.1 mL, 77.1 mmol, 3.0 eq). The resulting mixture was stirred for 1 h, then identical portions of lithium borohydride and MeOH were added. The resulting mixture was stirred at rt for 3 h. The reaction was monitored by TLC (5% MeOH, 95% EtOAc; detection: UV and ninhydrin, Rf=0.27. Note that the boronate co-eluted with the starting material, but after quenching, this spot disappeared). The reaction mixture was cooled to 0° C. and water was added very slowly (100-150 mL) to quench the reaction. On larger scales, the salts generated in the reaction were not completely soluble in the aqueous phase at this stage which complicated the extraction and led to lower yields. The resulting mixture was then stirred O/N. The aqueous phase was extracted with EtOAc (4×). The organic phase was dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. The compound was purified by flash chromatography (3% MeOH, 97% EtOAc) to give tether Ddz-T14 as a pale yellow solid (yield: 67%).
    
    


1H NMR (CDCl3, ppm): 7.53 (1H, s, RR′C═CH—S), 6.42-6.58 (2H, m, Ph), 6.35 (1H, t, Ph), 5.60-5.50 (1H, m, NH), 4.75 (2H, s, CH2OH), 4.60 (2H, d, CH2NHDdz), 3.78 (6H, s, 2×(CH3OPh)), 2.70-2.50 (1H, broad, OH), 1.76 (6H, s, RR′C(CH3)2).
13C NMR (CDCl3, ppm): 170, 161, 157, 156, 149, 116, 103, 99, 82, 61, 56, 42, 29.
Example T21
Standard Procedure for the Synthesis of Tether T21
See FIG. 6 for an outline of the synthetic scheme that provides the multi-step protocol for this tether containing methyl ether protection for its secondary hydroxyl groups. Alternative protection that is easier to remove, such as the acetonide, is also possible via this route.
Example T22
Standard Procedure for the Synthesis of Tether T22
An outline of the synthetic scheme that provides efficient routes to the diastereomeric forms of this tether is shown in FIG. 7.
Example T23
Standard Procedure for the Synthesis of Tether T23
The synthetic scheme that provides routes to this tether tether in shown is FIG. 8. Modifications can be used for homologous tethers.
Example T24
Standard Procedure for the Synthesis of Tether T24
The synthetic approach to this tether is shown in FIG. 9.
Example T24
Standard Procedure for the Synthesis of Tether T26
The synthetic scheme that provides this tether is shown in FIG. 10.
MW Calc. for C18H25NO6, 351.39; MS found (M+H)+ 352
Example T27
Standard Procedure for the Synthesis of Tether T27
An outline of the synthetic scheme is shown in FIG. 11.
Step T27-1:

    
    
        Ddz-N-(6-O-TBDPS, 2,3-deoxy-β-D-ribofuranosyl)methylamine (27-1). To a solution of 26-5 (20 g, 0.03 mmol) in EtOAc (40 mL) was added 10% rhodium on alumina (200 mg). The mixture was hydrogenated under atmospheric pressure using balloon of H2 gas. (CAUTION! Hydrogen gas is flammable.) After 12 h, the reaction mixture was filtered through a short pad of Celite® (World Minerals Inc., Santa Barbara, Calif.) and the filter cake washed with MeOH. The reaction had to be monitored by NMR since the starting material and product had the same Rf on TLC. The filtrate and washings were combined and concentrated under reduced pressure. The residue was azeotroped with dry toluene to afford a 98% yield of 27-1, which was used directly in the next step without further purification. MW Calc. for C34H45NO6Si, 591.8097; MS found (M+H)+ 592.
    
    


Step T27-2:
Ddz-N-(2,3-deoxy-β-D-ribofuranosyl)methylamine (Ddz-T27). The crude product, 27-1, from the previous step (100 g, 0.17 mol) was dissolved in anhydrous THF (500 mL). To the resulting clear solution was added TBAF (0.25 mol, 250 mL) and the reaction stirred for 2 h at rt. The reaction was monitored by TLC [(EtOAc/hexanes, 1:1,) detection: ninhydrin, Rf=0.5]. When the reaction was complete, the solution was poured into ice water and the aqueous solution was extracted with DCM (3×400 mL). The combined organic extract was washed with saturated citrate buffer (1×300 mL), H2O (200 mL) and brine (200 mL). The washed organic extract was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to give an oily residue. This residue was purified by flash chromatography (EtOAc/hexanes, 1:1, Rf=0.5) to give the protected tether (Ddz-T27) as a syrup (yield 90%).δ
1H NMR (CDCl3, 300 MHz): δ 1.61 (m, 1H), 1.74 (s, 6H); 1.80-1.88 (m, 3H); 2.66 (sb, 1H); 3.21 (m, 2H); 3.26 (m, 1H), 3.67 (m, 1H); 3.75 (s, 6H); 4.05 (m, 2H); 5.25 (m, 1H); 6.32 (m, 1H); 6.51 (m, 2H).
HPLC (Standard Gradient): Retention time (tr): 6.43 min
MW Calc. for C18H27NO6, 353.4101; MS found (M+H)+ 354.
Example T33
Standard Procedure for the Synthesis of Tether T33
An outline of the synthetic scheme towards this chiral tether is shown in FIG. 12. The enantiomers are accessed depending on the configuration of the starting lactic acid derivative with the (R)-isomer coming from (S)-methyl lactate and the (S)-isomer of T33 resulting from (R)-methyl lactate
1H NMR (CDCl3): δ 7.18-7.11 (m, 2H), 6.90 (m, 2H), 6.52 (m, 2H), 6.33 (m, 1H), 5.09 (bt, 1H), 4.52 (m, 1H), 3.77 (s, 6H), 3.08 (bq, 2H), 2.64 (bt, 2H), 1.75 (m, 8H); 1.27 (bd, 3H)
13C NMR (CDCl3): δ 160.8, 155.5, 149.5, 131.2, 130.6, 127.4, 121.2, 113.3, 103.2, 98.4, 80.7, 74.8, 66.5, 55.4, 40.2, 30.6, 29.3, 29.2, 27.4, 16.1
Example T38
Standard Procedure for the Synthesis of Tether T38
An outline of the synthetic scheme for racemic material is shown in FIG. 13. The enatiomers are accessed through the use of the optically pure propylene oxide enantiomers. Since the center of the epoxide is inverted during the protocol, the (R)-epoxide provides T38(S), while the (S)-epoxide provides T38(R).
1H NMR (CDCl3): δ 7.20-7.10, (m, 2H), 6.95-9.80 (m, 2H), 6.55 (bs, 2H), 6.35 (s, 1H), 5.18 (bt, 1H), 4.12 (m, 1H), 3.98 (m, 2H), 3.80 (s, 6H), 3.15 (bq, 2H), 2.65 (t, 2H), 1.98 (bs, 2H), 1.65 (bs, 6H), 1.25 (m, 3H).
Example T39
Standard Procedure for the Synthesis of Tether T39
See FIG. 14 for an outline of the synthetic scheme for racemic product. Enantiomeric versions can be accessed via resolution methodologies or use of an asymmetric Michael addition in the third step.
1H NMR (CDCl3): δ 7.11-7.08 (2H, m), 6.86 (1H, t), 6.76 (1H, d), 5.05 (1H, broad), 4.26-3.85 (4H, m), 3.22-3.07 (2H, m), 2.71 (1H, broad), 1.66-1.60 (2H, m), 1.33 (9H, s), 1.17 (3H, d).
13C NMR (CDCl3): δ 156.1, 135.0, 127.1, 127.0, 121.4, 111.7, 69.9, 61.5, 39.8, 38.4, 28.7, 20.7.
Example T40
Standard Procedure for the Synthesis of Tether T40
An outline of the synthetic scheme for racemic material is shown in FIG. 15, while FIG. 16 outlines the route to both enantiomers involving an enzymatic resolution as the key step.
1H NMR (CDCl3): δ 7.11-7.08 (2H, m), 6.86 (1H, t), 6.76 (1H, d), 5.05 (1H, broad), 4.26-3.85 (4H, m), 3.22-3.07 (2H, m), 2.71 (1H, broad), 1.66-1.60 (2H, m), 1.33 (9H, s), 1.17 (3H, d).
13C NMR (CDCl3): δ 156.1, 135.0, 127.1, 127.0, 121.4, 111.7, 69.9, 61.5, 39.8, 38.4, 28.7, 20.7.
Example T41
Standard Procedure for the Synthesis of Tether T41
See FIG. 18(a) for an outline of the synthetic scheme that provides an appropriately protected derivative for use in macrocycle construction via FIG. 1.
1H NMR (CDCl3): δ 1.23 (s, 3H), 1.49 (s, 3H), 1.69 (s, 3H), 1.74 (s, 3H), 1.90 (m, 2H), 2.35 (m, 1H), 3.35 (m, 2H), 3.76 (s, 6H), 3.92 (m, 2H), 4.40 (m, 2H), 5.10 (m, 1H), 6.15 (s, 1H), 6.25 (s, 2H).
13C NMR (CDCl3): δ 25.52 (CH3), 27.53 (CH3), 28.88 (CH3), 29.61 (CH3), 35.92 (CH2), 42.62 (CH2), 55.43 (CH3), 60.60 (CH2), 82.38 (CH), 83.33 (CH), 83.68 (CH), 84.96 (CH), 98.26 (CH), 103.23 (CH), 118.3 (Cq), 149.50 (Cq), 156.20 (Cq), 160, 02 (Cq)
MW Calcd. for C22H33NO8: 439.50; MS Found: (M+H)+ 440
Example T54
Standard Procedure for the Synthesis of Tether T54
See FIG. 18(c) for an outline of the synthetic scheme from a T55 derivative.
1H NMR (CDCl3): δ 1.55 (m, 2H), 1.72 (s, 6H), 1.8-2.01 (m, 4H), 2.75 (sb, 1H), 3.10 (m, 1H), 3.32 (m, 1H), 3.65 (s, 6H), 3.66 (m, 2H), 3.90-4.01 (m, 2H), 5.30 (m, 1H), 6.30 (s, 1H), 6.50 (s, 2H).
13C NMR (CDCl3): δ 28.04 (CH2), 29.18 (CH3), 29.34 (CH3), 31.69 (CH2), 38.08 (CH2), 44.94 (CH2), 55.41 (CH3), 61.28 (CH2), 78.84 (CH), 79.41 (CH), 80.75 (Cq), 98.44 (CH), 103.15 (CH), 149.44 (Cq), 155.64 (Cq), 160.81 (Cq). MW Calcd. for C19H29NO6: 367.44; MS Found: (M+H)+ 368
Example T55
Standard Procedure for the Synthesis of Tether T55
See FIG. 18(b) for an outline of the synthetic scheme.
1H NMR (CDCl3): δ 1.66 (s, 3H), 1.71 (s, 3H), 1.82 (m, 1H), 1.89 (m, 1H), 3.26 (m, 2H), 3.77 (s, 6H), 3.80 (m, 2H), 4.84 (m, 1H), 4.95 (m, 1H), 5.20 (m, 1H), 5.70 (m, 1H), 5.85 (m, 1H), 6.32 (s, 1H), 6.49 (s, 2H).
13C NMR (CDCl3): δ 29.06 (CH3), 29.42 (CH3), 38.73 (CH2), 44.87 (CH2), 55.45 (CH3), 61.01 (CH2), 80.77 (Cq), 85.84 (CH), 86.25 (CH), 98.28 (CH), 103.28 (CH), 127.84 (CH), 131.95 (CH), 149.42 (Cq), 155.59 (Cq), 160.79 (Cq).
MW Calcd. for C19H27NO6: 365.42; MS Found: (M+H)+ 366
Example T56
Standard Procedure for the Synthesis of Precursor (56-1) for Tethers T56 and T57
For some of the tether structures, specifically those arising from the ring-closing metathesis methodology (RCM, FIG. 2), the tether is not added as an already assembled unit, but is constructed during the macrocyclization reaction from appropriate precursor pieces. One such example id shown in FIG. 19 in which 56-1, containing a pendant alkene moiety, will be subjected to RCM whereby the alkene will join with an alkene in another part of the substrate to form the macrocyclic ring and, hence, construct tether T56 (or homologues). Reduction of the double bond in macrocycles containing T56 leads to macrocycles containing T57. Other tethers that were constructed in this manner include T46, T47, T49, and T51.
Table 1 lists the structural features for 60 preferred embodiments of compounds of formula (I).
Table 2 gives the Mass Spectrum analytical data for these compounds.







TABLE 1







Representative Compounds of formula (I)








(I)











Cmpd
A1
A2
A3
T*














201












T40(S)


 


202












T40(S)


 


203












T38(S)


 


204












T40(R)


 


205












T40(R)


 


206












T38(R)


 


207












T40(S)


 


208












T38(S)


 


209












T40


 


210












T39


 


211












T40(R)


 


212












T58


 


213












T39


 


214












T40(R)


 


215












T59(S)


 


216












T59(R)


 


217












T38(R)


 


218












T59(S)


 


219












T59(R)


 


220












T33(R)


 


222












T38


 


223












T38


 


224












T41


 


225












T41


 


226












T33(S)


 


227












T33(R)


 


228












T12


 


229












T56(Y = H)


 


230












T57(Y = H)


 


231












T56(Y = Me)


 


232












T57(Y = Me)


 


233












T21(Y = H)


 


234












T26


 


235












T12


 


236












T13


 


237












T14


 


238












T12


 


241












T38


 


242












T33(R)


 


243












T33(S)


 


244












T33(R)


 


245












T33(S)


 


246












T39


 


247












T58


 


248












T40


 


249












T21(Y = H)


 


250












T24


 


251












T12


 


252












T27


 


253












T14


 


254












T33(R)


 


255












T33(S)


 


256












T39


 


257












T40


 


258












T58


 


259












T12


 


260












T46


 


261












T47


 


262












T49


 


263












T51


 


264












T51





W is NH except for compounds 229 to 232 where W is O.


*Designation in parentheses indicates the absolute configuration (R or S) of the chiral center on the tether. If no configuration is so designated, the center is racemic. Other designations indicate the identity of a variable substituent.













TABLE 2







Mass Spectral Analyses for Representative Compounds of formula I













Molecular
Monoisotopic
MS Found


Cmpd
Molecular Formula
Weight
Mass
(M + H)+














201
C31H42N7O4F3
633.7
633
634


202
C31H44N5O4F
569.7
569
570


203
C30H42N7O4Cl
600.2
599
600


204
C31H42N7O4F3
633.7
633
634


205
C31H44N5O4F
569.7
569
570


206
C30H42N7O4Cl
600.2
599
600


207
C32H43N4O4Cl
583.2
582
583


208
C32H43N4O4F
566.7
566
567


209
C32H43N4O4Cl
583.2
582
583


210
C31H43N4O4Cl
571.2
570
571


211
C32H43N4O4Cl
583.2
582
583


212
C33H45N4O4Cl
597.2
596
597


213
C31H43N4O4F
554.7
554
555


214
C32H43N4O4F
566.7
566
567


215
C31H41N4O5Cl
585.1
584
585


216
C31H41N4O5Cl
585.1
584
585


217
C32H43N4O4F
566.7
566
567


218
C31H41N4O5F
568.7
568
569


219
C31H41N4O5F
568.7
568
569


220
C32H43N4O4F
566.7
566
567


222
C32H46N4O5
566.7
566
567


223
C32H43N4O4Cl
583.2
582
583


224
C27H39N4O6Cl
551.1
550
551


225
C27H42N4O7
534.6
534
535


226
C31H44N4O5
552.7
552
553


227
C31H44N4O5
552.7
552
553


228
C30H38N6O3S
562.7
562
563


229
C28H41N3O8
547.6
547
548


230
C28H43N3O8
549.7
549
550


231
C30H45N3O8
575.7
575
576


232
C30H47N3O8
577.7
577
578


233
C25H38N4O7
506.6
506
507


234
C25H36N4O5
472.6
472
473


235
C38H42N4O4S
650.8
650
651


236
C24H33N5O5
471.5
471
472


237
C24H33N5O4S
487.6
487
488


238
C33H40N4O4S
588.8
588
589


241
C30H39N4O4F
538.7
538
539


242
C31H44N4O4
536.7
536
537


243
C31H44N4O4
536.7
536
537


244
C30H39N4O4F
538.7
538
539


245
C30H39N4O4F
538.7
538
539


246
C30H39N4O4F
538.7
538
539


247
C31H41N4O4F
552.7
552
553


248
C30H39N4O4F
538.7
538
539


249
C24H33N4O6F
492.5
492
493


250
C26H41N4O3F
476.6
476
477


251
C31H36N4O3S
544.7
544
545


252
C23H34N4O4
430.5
430
431


253
C22H29N5O3S
443.6
443
444


254
C33H45N4O4Cl
597.2
596
597


255
C33H45N4O4Cl
597.2
596
597


256
C33H45N4O4Cl
597.2
596
597


257
C33H45N4O4Cl
597.2
596
597


258
C34H47N4O4Cl
611.2
611
612


259
C35H42N4O3S
598.8
598
599


260
C23H35N4O3F
434.5
434
435


261
C26H39N4O3Cl
491.1
490
491


262
C27H41N4O3Cl
505.1
504
505


263
C28H43N4O3Cl
519.1
518
519


264
C29H45N4O3Cl
533.1
532
533





Notes


1. Molecular formulas and molecular weights (MW) are calculated automatically from the structure via ActivityBase ® software (ID Business Solution, Ltd., Guildford, Surrey, UK) or, for MW only, from the freeware program Molecular Weight Calculator v. 6.32


2. M + H obtained from LC-MS analysis


3. All analyses conducted on material after preparative purification






Biological Evaluation for Compounds of the Invention
The compounds of the present invention were evaluated for their ability to interact at the human motilin receptor and the human ghrelin receptor utilizing competitive radioligand binding assays as described in Method B1 and B2, respectively. Further characterization of the interaction can be performed utilizing the functional assays described in Methods B3 and B4 for the motilin and ghrelin receptors, respectively. All of these methods can be conducted, if so desired, in a high throughput manner to permit the simultaneous evaluation of many compounds.
Results for the examination of representative compounds of the present invention using Methods B1 and B2 are presented in Table 3.
Example Method B1
Competitive Radioligand Binding Assay (Motilin Receptor)
Materials:

    
    
        Membranes were prepared from CHO cells stably transfected with the human motilin receptor and utilized at a quantity of 1.5 μg/assay point. [PerkinElmerTM SignalScreen® Product #6110544, PerkinElmer, Inc. Wellesley, Mass.]
        [125I]-Motilin (PerkinElmer, #NEX-378); final concentration: 0.04-0.06 nM
        Motilin (BachemTM, #H-4385, Bachem Bioscience Inc., King of Prussia, Pa.); final concentration: 1 μM
        Multiscreen® Harvest plates-GF/B (MilliporeTM, #MAHFB1H60, Billerica, Mass.)
        Deep-well polypropylene titer plate (Beckman CoulterTM, #267006, Fullerton, Calif.)
        TopSeal-ATM (PerkinElmer, #6005185, Wellesley, Mass.)
        Bottom seal (Millipore, #MATAH0P00)
        MicroScint-0TM (PerkinElmer, #6013611, Wellesley, Mass.)
        Binding Buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA, 0.1% BSA
    
    


Assay Volumes:

    
    
        150 μL of membranes diluted in binding buffer
        10 μL of compound diluted in binding buffer
        10 μL of radioligand ([125I]-Motilin) diluted in binding buffer
    
    


Final Test Concentrations (N=11) for Compounds:

    
    
        10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 μM.
    
    


Compound Handling:
Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at −20° C. until the day of testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.5%.
Assay Protocol:
In deep-well plates, diluted cell membranes (1.5 μg/mL) are combined with 10 μL of either binding buffer (total binding, N=5), 1 μM motilin (non-specific binding, N=3) or the appropriate concentration of test compound. The reaction is initiated by addition of 10 μl of [125I]-motilin (final conc. 0.04-0.06 nM) to each well. Plates are sealed with TopSeal-ATM, vortexed gently and incubated at room temperature for 2 hours. The reaction is arrested by filtering samples through pre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen® Harvest plates using a Tomtec Harvester (Tomtec, Hamden, Conn.), washed 9 times with 500 μL of cold 50 mM Tris-HCl (pH 7.4), and than plates are air-dried in a fumehood for 30 minutes. A bottom seal is applied to the plates prior to the addition of 25 μL of MicroScint-0® to each well. Plates are then sealed with TopSeal-ATM and counted for 30 sec per well on a TopCount® Microplate Scintillation and Luminescence Counter (PerkinElmer, Wellesley, Mass.) where results are expressed as counts per minute (cpm).
Data are analyzed by GraphPadTM Prism® (GraphPad Software, San Diego, Calif.) using a variable slope non-linear regression analysis. Ki values were calculated using a Kd value of 0.16 nM for [125I]-motilin (previously determined during membrane characterization).




  
    D
    max
  
  =
  
    1
    -
    
      
        
          
            
              
                
                  test
                  ⁢
                  
                    
                  
                  ⁢
                  concentration
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  with
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  maximal
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  displacement
                
                -
              
            
          
          
            
              
                non
                ⁢
                
                  -
                
                ⁢
                specific
                ⁢
                
                  
                
                ⁢
                binding
              
            
          
        
        
          
            total
            ⁢
            
              
                
              
              ⁢
              
                
              
            
            ⁢
            binding
          
          -
          
            non
            ⁢
            
              -
            
            ⁢
            specific
            ⁢
            
              
            
            ⁢
            binding
          
        
      
      ×
      100
    
  




where total and non-specific binding represent the cpm obtained in the absence or presence of 1 μM motilin, respectively.
Example Method B2
Competitive Radioligand Binding Assay (Ghrelin Receptor)
The competitive binding assay at the human growth hormone secretagogue receptor (hGHS-R1a) was carried out analogously to assays described in the literature. (Bednarek M A et al. (2000), Structure-function studies on the new growth hormone-releasing peptide ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a; J Med Chem 43:4370-4376.
Palucki B L et al. (2001), Spiro(indoline-3,4′-piperidine) growth hormone secretagogues as ghrelin mimetics; Bioorg Med Chem Lett 11:1955-1957.)
Materials

    
    
        Membranes (GHS-R/HEK 293) were prepared from HEK-293 cells stably transfected with the human ghrelin receptor (hGHS-R1a). These membranes were provided by PerkinElmer BioSignal® (#RBHGHSM, lot#1887, PerkinElmer, Wellesley, Mass.) and utilized at a quantity of 0.71 μg/assay point.
        [125I]-Ghrelin (PerkinElmer, #NEX-388); final concentration: 0.0070-0.0085 nM
        Ghrelin (Bachem, #H-4864, Bachem Bioscience Inc., King of Prussia, Pa.); final concentration: 1 μM
        Multiscreen® Harvest plates-GF/C (Millipore, #MAHFC1H60, Billerica, Mass.)
        Deep-well polypropylene titer plate (Beckman Coulter, #267006, Fullerton, Calif.)
        TopSeal-ATM (PerkinElmer, #6005185, Wellesley, Mass.)
        Bottom seal (Millipore, #MATAH0P00)
        MicroScint-0TM (PerkinElmer, #6013611, Wellesley, Mass.)
        Binding Buffer: 25 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM MgCl2, 2.5 mM EDTA, 0.4% BSA
    
    


Assay Volumes
Competition experiments were performed in a 300 μL filtration assay format.

    
    
        220 μL of membranes diluted in binding buffer
        40 μL of compound diluted in binding buffer
        40 μL of radioligand ([125I]-Ghrelin) diluted in binding buffer
    
    


Final test concentrations (N=1) for compounds of the present invention:
10, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, 0.001 μM.
Compound Handling
Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at −80° C. until the day of testing. On the test day, compounds were allowed to thaw at rt overnight and then diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximal final DMSO concentration in the assay was 0.1%.
Assay Protocol
In deep-well plates, 220 μL of diluted cell membranes (final concentration: 0.71 μg/well) were combined with 40 μL of either binding buffer (total binding, N=5), 1 μM ghrelin (non-specific binding, N=3) or the appropriate concentration of test compound (N=2 for each test concentration). The reaction was initiated by addition of 40 μl, 1, of [125I]-ghrelin (final conc. 0.0070-0.0085 nM) to each well. Plates were sealed with TopSeal-ATM, vortexed gently and incubated at rt for 30 min. The reaction was arrested by filtering samples through Multiscreen® Harvest plates (pre-soaked in 0.5% polyethyleneimine) using a Tomtec® Harvester (Tomtec, Hamden, Conn.), washed 9 times with 500 μL of cold 50 mM Tris-HCl (pH 7.4, 4° C.), and then plates were air-dried in a fumehood for 30 min. A bottom seal was applied to the plates prior to the addition of 25 μL of MicroScint-0TM to each well. Plates were than sealed with TopSeal-ATM and counted for 30 sec per well on a TopCount® Microplate Scintillation and Luminescence Counter (PerkinElmer, Wellesley, Mass.) using a count delay of 60 sec. Results were expressed as counts per minute (cpm).
Data were analyzed by GraphPadTM Prism® (GraphPad Software, San Diego, Calif.) using a variable slope non-linear regression analysis. Ki values were calculated using a Kd value of 0.01 nM for [125I]-ghrelin (previously determined during membrane characterization).
Dmax values were calculated using the following formula:




  
    D
    max
  
  =
  
    1
    -
    
      
        
          
            
              
                
                  test
                  ⁢
                  
                    
                  
                  ⁢
                  concentration
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  with
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  maximal
                  ⁢
                  
                    
                      
                    
                    ⁢
                    
                      
                    
                  
                  ⁢
                  displacement
                
                -
              
            
          
          
            
              
                non
                ⁢
                
                  -
                
                ⁢
                specific
                ⁢
                
                  
                
                ⁢
                binding
              
            
          
        
        
          
            total
            ⁢
            
              
                
              
              ⁢
              
                
              
            
            ⁢
            binding
          
          -
          
            non
            ⁢
            
              -
            
            ⁢
            specific
            ⁢
            
              
            
            ⁢
            binding
          
        
      
      ×
      100
    
  




where total and non-specific binding represent the cpm obtained in the absence or presence of 1 μM ghrelin, respectively.
Example Method B3
Aequorin Functional Assay (Motilin Receptor)
Materials:

    
    
        Membranes were prepared using AequoScreenTM (EUROSCREEN, Belgium) cell lines expressing the human motilin receptor (cell line ES-380-A; receptor accession #AF034632). This cell line is constructed by transfection of the human motilin receptor into CHO-K1 cells co-expressing Gα16 and the mitochondrially targeted Aequorin (Ref #ES-WT-A5).
        Motilin (Bachem, #H-4385, Bachem Bioscience Inc. King of Prussia, Pa.)
        Assay buffer: DMEM-F12 (Dulbeccoe's Modified Eagles Medium) with 15 mM HEPES and 0.1% BSA (pH 7.0)
        Coelenterazine (Molecular ProbesTM, Leiden, The Netherlands)
    
    


Final Test Concentrations (N=5) for Compounds:

    
    
        10, 3.16, 1, 0.316, 0.1 μM.
    
    


Compound Handling:
Compounds were provided as dry films at a quantity of approximately 1.2 μmol in pre-formatted 96-well plates. Compounds were dissolved in 100% DMSO at a concentration of 10 mM and stored at −20° C. until further use. Daughter plates were prepared at a concentration of 500 μM in 30% DMSO with 0.1% BSA and stored at −20° C. until testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.6%.
Cell Preparation:
Cells are collected from culture plates with Ca2+ and Mg2+-free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 minutes at 1000×g, resuspended in assay buffer (see above) at a density of 5×106 cells/mL and incubated overnight in the presence of 5 μM coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5×105 cells/mL.
Assay Protocol:
For agonist testing, 50 μl of the cell suspension was mixed with 50 μl of the appropriate concentration of test compound or motilin (reference agonist) in 96-well plates (duplicate samples). The emission of light resulting from receptor activation was recorded using the Functional Drug Screening System 6000 ‘FDSS 6000’ (Hamamatsu Photonics K.K., Japan).
For antagonist testing, an approximate EC80 concentration of motilin (i.e. 0.5 nM; 100 μL) was injected onto the cell suspension containing the test compounds (duplicate samples) 15-30 minutes after the end of agonist testing and the consequent emission of light resulting from receptor activation was measured as described in the paragraph above.
Results are expressed as Relative Light Units (RLU). Concentration response curves were analyzed using GraphPadTMPrism® (GraphPad Software, San Diego, Calif.) by non-linear regression analysis (sigmoidal dose-response) based on the equation E=Emax/(1+EC50/C)n where E is the measured RLU value at a given agonist concentration (C), Emax is the maximal response, EC50 is the concentration producing 50% stimulation and n is the slope index. For agonist testing, results for each concentration of test compound were expressed as percent activation relative to the signal induced by motilin at a concentration equal to the EC80 (i.e. 0.5 nM). For antagonist testing, results for each concentration of test compound were expressed as percent inhibition relative to the signal induced by motilin at a concentration equal to the EC80 (i.e. 0.5 nM).
Example Method B4
Aequorin Functional Assay (Ghrelin Receptor)
Materials

    
    
        Membranes were prepared using AequoScreenTM (EUROSCREEN, Belgium) cell lines expressing the human ghrelin receptor (cell line ES-410-A; receptor accession #60179). This cell line is constructed by transfection of the human ghrelin receptor into CHO-K1 cells co-expressing G□16 and the mitochondrially targeted Aequorin (Ref #ES-WT-A5).
        Ghrelin (reference agonist; Bachem, #H-4864, Bachem Bioscience Inc., King of Prussia, Pa.)
        Assay buffer: DMEM (Dulbecco's Modified Eagles Medium) containing 0.1% BSA (bovine serum albumin; pH 7.0.
        Coelenterazine (Molecular Probes, Leiden, The Netherlands)
    
    


Final test concentrations (N=8) for compounds of the invention:
10, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001 μM.
Compound Handling
Stock solutions of compounds (10 mM in 100% DMSO) were provided frozen on dry ice and stored at −20° C. prior to use. From the stock solution, mother solutions were made at a concentration of 500 μM by 20-fold dilution in 26% DMSO. Assay plates were then prepared by appropriate dilution in DMEM medium containing 0.1% BSA. Under these conditions, the maximal final DMSO concentration in the assay was <0.6%.
Cell Preparation
AequoScreenTM cells were collected from culture plates with Ca2+ and Mg2+-free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 min at 1000×g, re-suspended in DMEM-Ham's F12 containing 0.1% BSA at a density of 5×106 cells/mL, and incubated overnight at rt in the presence of 5 μM coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5×105 cells/mL.
Assay Protocol
For agonist testing, 50 μL of the cell suspension was mixed with 50 μL of the appropriate concentration of test compound or ghrelin (reference agonist) in 96-well plates (duplicate samples). Ghrelin (reference agonist) was tested at several concentrations concurrently with the test compounds in order to validate the experiment. The emission of light resulting from receptor activation in response to ghrelin or test compounds was recorded using the Hamamatsu FDSS 6000 reader (Hamamatsu Photonics K.K., Japan).
Analysis and Expression of Results
Results were expressed as Relative Light Units (RLU). Concentration response curves were analyzed using GraphPad Prism (GraphPad Software, San Diego, Calif.) by non-linear regression analysis (sigmoidal dose-response) based on the equation E=Emax/(1+EC50/C)n where E was the measured RLU value at a given agonist concentration (C), Emax was the maximal response, EC50 was the concentration producing 50% stimulation and n was the slope index. For agonist testing, results for each concentration of test compound are expressed as percent activation relative to the signal induced by ghrelin at a concentration equal to the EC80 (i.e. 3.7 nM). EC50, Hill slope and % Emax values are reported.







TABLE 3







Biological Activity of Representative Compounds of formula I












Binding Affinity [Ki




Compound
(μM)]1
Receptor2






201
A
motilin (human)



202
A
motilin (human)



203
A
motilin (human)



204
A
motilin (human)



205
B
motilin (human)



206
B
motilin (human)



207
A
motilin (human)



208
A
motilin (human)



209
A
motilin (human)



210
A
motilin (human)



211
A
motilin (human)



212
A
motilin (human)



213
A
motilin (human)



214
A
motilin (human)



215
A
motilin (human)



216
A
motilin (human)



217
B
motilin (human)



218
B
motilin (human)



219
B
motilin (human)



220
B
motilin (human)



221
B
motilin (human)



222
A
motilin (human)



223
A
motilin (human)



224
B
motilin (human)



226
B
motilin (human)



227
B
motilin (human)



228
B
motilin (human)



235
C
motilin (human)



236
B
motilin (human)



237
B
motilin (human)



241
A
ghrelin (human)



242
A
ghrelin (human)



243
A
ghrelin (human)



244
A
ghrelin (human)



245
A
ghrelin (human)



246
B
ghrelin (human)



247
B
ghrelin (human)



248
B
ghrelin (human)



251
B
ghrelin (human)



254
A
ghrelin (human)



255
A
ghrelin (human)



256
B
ghrelin (human)



257
A
ghrelin (human)



258
B
ghrelin (human)



259
C
ghrelin (human)



260
C
ghrelin (human)



261
C
ghrelin (human)



262
B
ghrelin (human)



263
B
ghrelin (human)



264
B
ghrelin (human)





1Activity presented indicated in the following ranges: A = 0.001-0.10 μM, B = 0.1-1.0 μM, C = 1.0-10.0 μM


2Binding conducted using the Standard Methods described in the Examples






Although preferred embodiments of the present invention have been described in detail herein, it is to be understood that the invention is not limited to these precise embodiments and that various changes and modifications may be effected therein without departing from the scope or spirit of the present invention.

## Claims

1. A compound of formula W-T-X, wherein T is selected from the group consisting of




wherein:
W is selected from the group consisting of —OH, —NH2 and —NHR3, wherein R3 is unsubstituted methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, n-butyl, isobutyl or sec-butyl, and X is —NH2 or —NHR3, wherein R3 is unsubstituted methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, n-butyl, isobutyl or sec-butyl;
(W) indicates the point of attachment of T to W; and (X) indicates the point of attachment of T to X, and
W and/or X may be optionally protected by one or more protecting groups.

2. The compound of claim 1, wherein R3 is methyl, ethyl, propyl or isopropyl.

3. The compound of claim 1, wherein W and/or X is protected by one or more protecting groups selected from the group consisting of tert-butoxycarbonyl (Boc), dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz), and 9-fluorenylmethoxycarbonyl (Fmoc).

4. The compound of claim 1, wherein T is the following:

5. The compound of claim 1, wherein T is the following: